Apparatus and method for growing anaerobic microorganisms

ABSTRACT

An apparatus for growing anaerobic microorganisms is provided having a dish top that contains a sealing ring upon which the media surface in the dish bottom rests when the apparatus is inverted. The contact between the sealing ring and the media surface forms a seal that traps the gas in the headspace between the media surface and the inside of the dish top. A oxygen reducing agent can also be incorporated into the media together, in some instances, with a substrate which react with oxygen in the media and with oxygen in the headspace thereby creating an environment suitable for growing anaerobic, microaerophilic and facultative anaerobic microorganisms.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part application of Ser.No. 08/963/664, filed Nov. 3, 1997, now U.S. Patent No., which is acontinuation application of Ser. No. 08/237,773, filed May 4, 1994, nowU.S. Pat. No. 5,830,746, issued Nov. 3, 1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of Invention

[0003] The present invention relates to an apparatus and method forgrowing anaerobic microorganisms. The apparatus is comprised of aspecially designed culture dish which can be reconfigured such as byinverting the dish to produce an anaerobic environment. An oxygenreducing agent such as a biocatalytic oxygen reducing agent can also beincorporated into the media present in the apparatus together, in somecircumstances, with a substrate. The biocatalytic oxygen reducing agentand the substrate present in the media react with oxygen enclosed in theculture dish to create an environment suitable for growing andmaintaining anaerobic microorganisms.

[0004] 2. Description of Prior Art

[0005] Microorganisms are important to our well being. This is evidentin health care, agriculture and industry. To be able to simply andquickly isolate and grow microbes is economically important. Forexample, being able to quickly and specifically isolate and identify amicrobe responsible for infection is important in the human health carefield. This basic technique is also important in the agricultureindustry. Large scale processing of food requires constant microbialmonitoring. The speed and efficiency at which this can be donedetermines the length of time finished food products must be held instorage before they can be distributed for sale.

[0006] Control of the environment is necessary for control of microbialgrowth. In particular, control of oxygen content in the immediateenvironment is crucial for microbial growth. Microorganisms can bedivided into groups based on their need for, and tolerance of, oxygen.There are those that require oxygen to grow. These are “aerobes”. Somemicroorganisms are able to grow with or without oxygen. These are“facultative anaerobes”. Another group of microorganisms can grow onlyin the presence of very low levels of oxygen. These are the“microaerophiles”. Finally, some microorganisms can not tolerate oxygen.They are inhibited by it or may be killed by it. These are the“anaerobes”.

[0007] This fundamental property of microorganisms, that is theirability to grow in or tolerate oxygen, is used daily to isolate, grow,and manipulate them. One basic technique in microbiology, is the platingmethod. This generally involves the use of a dish, developed by Petri(i.e. “Petri dish”) in 1880's, and solidified (agar or gelatin-based)medium.

[0008] A Petri dish is usually a round, shallow, flat-bottomed, glass orplastic dish (often e.g. 10 cm diameter) with a vertical side, thatcooperates with a similar, slightly larger structure which forms aloosely-fitting lid. Petri dishes are used in microbiology, e.g., forthe preparation of plates.

[0009] The purpose of the Petri dish is to provide a controlledenvironment for selectively growing microbes. The dish is sterilized anddesigned to maintain a sterile environment inside while freelyexchanging gases, normally air, with the outside environment.

[0010] The medium utilized in conjunction with the Petri dish can beformulated to provide a necessary and selective environment for aspecific microorganism. Solid medium in a Petri dish can be preparedusing aseptic technique by pouring sterile molten or liquid (agar- orgelatin-based) medium into a Petri dish to a depth of 3-5 mm andallowing it to set. Generally, freshly poured plates to be used forseparation and/or generation of microbes should be left for 30 minutesin a 45° C. hot-air incubator with the lid partly off so that thesurface moisture can evaporate. Such “drying” before inoculationprevents unwanted spreading of the inoculum in the surface film of themoisture.

[0011] The solid medium surface inside the dish provides a place to growmicroorganisms. By inoculating (or “plating”) the surface of the agar ina controlled way (i.e. “streaking”), single colonies of a microorganismcan be obtained. With this technique the microbiologist can separatemicrobes one from another. Isolation and purification is mandatory tofurther characterization and study. Using this dish design, amicrobiologist can isolate and grow the great majority of microorganismsknown today.

[0012] Working with microbes that are microaerophiles or anaerobes posesa problem. The culture dishes for these microbes must be incubated in acontrolled gaseous environment that lacks oxygen, or at least most ofthe oxygen, found in air. This is done by placing the culture or Petridish containing medium inside a container that is sealed from theoutside atmosphere. For one or a few dishes, a sealable bag or jar(i.e., “Brewer Jar”) is used (Becton Dickinson Microbiology Systems,1994 Catalog, p 89 p 94). In this case, chemicals and a catalyst (seeU.S. Pat. No. 4,287,306 issued Sep. 1, 1982 to Brewer entitled“Apparatus for Generation of Anaerobic Atmosphere”) are placed insidethe container that, when activated chemically, reacts with the oxygen inthe container, thus removing it. The catalyst is necessary to bringabout the reaction at low temperatures in a short time.

[0013] In addition, for many culture dishes, a sealed table-top chambercan be used (Anaerobe Systems, San Jose, Calif.). This chamber isevacuated and flushed with inert gases, such as nitrogen and/or carbondioxide. Sometimes chemicals and a catalyst are used to consume theoxygen inside the chamber and fresh, inert gas is supplied as needed.The microbiologist works with the culture dishes inside of this chamberthrough ports fitted with gloves. A means is provided for introducingmaterials into and taking items out of the chamber without breaching theanaerobic environment inside.

[0014] Work with microaerophiles and anaerobes under these conditions islabor intensive, difficult, expensive, and time consuming. Themicrobiologist is often frustrated by having to wait for the slowestgrowing microbe in order to retrieve all culture dishes from a bag orjar since once the bag or jar is opened, the microbes are exposed tooxygen. A failure in the system can be catastrophic for all of themicrobial isolates inside.

[0015] To overcome many of these problems (see U.S. Pat. No. 2,348,448issued May 9, 1944 to Brewer entitled “Apparatus for the Cultivation ofAnaerobic and Microaerophilic Organisms”) Brewer developed a culturedish lid (i.e., “Brewer Lid”) that formed a seal between a ring insidethe lid with the agar or gelatin-based surface. Within the dish, a verysmall, defined headspace is formed by the lid and the agar surface. Ananaerobic environment is created inside this trapped headspace byreacting oxygen with chemical reducing agents, such as thioglycollate,incorporated in the medium. The limited volume of the headspace isimportant to the function of the Brewer Lid.

[0016] However, a number of drawbacks exist in the use of the BrewerLid. The capacity and the rate for oxygen removal is limited by thesensitivity of the microorganism to the chemical reducing agent in themedium (see “Mechanism of Growth Inhibitory Effect of cysteine onEscherichia coli.” of Kari, et al., J. Gen. Microbiol., 68, 1971, p. 349and “Methods for General and Molecular Bacteriology”, Editor: Gerherdt,American Society for Microbiology, 1994, p. 146.). Moreover, the lid ismade of heavy glass and is expensive. It is available today (KimbleGlass Company, Vineland, N.J.), but is not widely used because ofserious limitations that include cost, handling difficulties, and poorresponse of anaerobic microorganisms.

[0017] Another limitation is caused by the material of construction. Theglass Brewer Lid is made very heavy to insure a good seal between thering inside the Brewer Lid and the agar surface. Cultures dish bottomsfitted with the heavy Brewer Lid are not easy to handle or to moveabout. They can not be stacked inside an incubator. Thus, preciousincubator space is wasted. Stacked dishes crush the agar medium of thelowest dishes in the stack, because of the weight of the dishes abovethem. This causes the headspace above the agar to collapse resulting incontact between the inside of the Brewer Lid and the agar surface. Whenthis happens, the microbial growth on the surface is spread out andseparation of individual colonies is lost. Motile microbes will migrateand further frustrate separation.

[0018] Because of their weight and material of construction, Brewer Lidsdo not lend themselves to commercial production of pre-made agar orgelatin-based plates. The commercial process requires assembly linefilling of the dishes, packaging the filled dishes in stacks, andhandling and storing these dishes. Pre-made agar plates are widely usedin clinical microbiological laboratories. This limitation of the BrewerLid is economically significant.

[0019] The headspace inside the Brewer Lid formed by the lid and agarsurface is very small. This limited headspace is determined by theability of the chemical reducing agent (H₂S, cysteine, thioglycollate,etc.) to reduce oxygen in the headspace. The amount of chemical reducingagent used in the medium in turn is constrained by anaerobicmicroorganism's sensitivity to it. The sum of these limitations is avery small head space that imparts severe problems to the function ofthe Brewer Lid for its intended purpose, i.e. to grow anaerobic andmicroaerophilic microorganisms.

[0020] Another limitation of the Brewer Lid is that the very limitedhead space can not hold much moisture. Fresh agar medium is generallygreater than 98 percent water. Upon incubation, water in the mediumevaporates and condenses upon the upper surface of the inside of thelid. This condensate can become sufficient to fall to the agar surfaceand to flood it. Under such conditions, the plate is ruined and can notbe used for isolation and purification of the microbe.

[0021] The very limited headspace imposes still more limitations on theBrewer Lid. No provision is made to incorporate CO₂ into the headspaceabove the agar surface. This is important for the rapid growth of somemicroorganisms and may be required by others. Yet this feature should bemade optional for the microbiologist, because for some uses of theculture dish the microbiologist may not want to include CO₂ in theheadspace. Reports show that CO₂ can change the pH of the medium itcontacts. This in turn can interfere with the determination ofsusceptibility to some antibiotics (see “Effect of CO₂ onSusceptibilities of Anaerobes to Erythromycin, Azithromycin,Clarithromycin, and Roxithromycin”, Spangler, et al., Antimicrob. AgentsChemotherapy, 38, p. 20, 1994). Since CO₂ is generated in anaerobic jarsand bags by commercial catalysts products, this problem is commonlyencountered. CO₂ is a component of the gas used to flush anaerobicchambers and incubators too.

[0022] Another desired feature for a self contained culture dish is anindicator to show that the headspace is anaerobic. These features aredifficult to impossible to include in the Brewer Lid because of the verysmall space between inside the lid top and the agar surface.

[0023] Several attempts have been made to design a culture dish thatprovides a self-contained environment for growing anaerobicmicroorganisms (see U.S. Pat. No. 2,701,229 issued Feb. 1, 1955 toScherr entitled “Apparatus for the Cultivation of Microorganisms”; U.S.Pat. No. 3,165,450 issued Jan. 12, 1965 to Scheidt entitled “AnaerobicCulturing Device”; U.S. Pat. No. 4,294,924 issued Oct. 13, 1981 toPepicelli, et al. entitled “Method and Container for Growth of AnaerobicMicroorganisms”; U.S. Pat. No. 4,299,921 issued Nov. 10, 1981 to Youssefentitled “Prolonged Incubation Microbiological Apparatus and FilterGaskets Thereof”; and U.S. Pat. No. 4,859,586 issued Aug. 8, 1989 toEisenberg entitled “Device for Cultivating Bacteria”). The fact that theBrewer Lid and none of these inventions are commonly or commerciallyavailable or used widely by microbiologists today, attest to theirlimitations and shortcomings. The need to simplify and reduce the costfor isolating and growing anaerobic and microaerophilic microorganismsstill exists today.

[0024] It is therefore an object of the present invention to provide animproved apparatus and method for cultivating and/or enumeratinganaerobic microorganisms which obviate the above-mentioned disadvantagesof the prior art.

[0025] Another object of the present invention is to provide an improvedanaerobic culturing apparatus which is extremely simple, inexpensive andeasy to use and wherein the proper anaerobic environment is produced andmaintained in an extremely efficient manner.

[0026] These and other additional objects and advantages of the presentinvention will become apparent from the following description of theinvention.

SUMMARY OF THE INVENTION

[0027] The present inventors have designed a novel culture apparatus ordish in order to eliminate many of the difficulties observed in theprior art. It has been found that the use of the new culture dish (i.e.,“OxyDish™”) together with an oxygen reducing agent (preferably abiocatalytic oxygen reducing agent) and, in some instances, a substrate,produces a controlled, self-contained environment for isolating,enumerating, identifying and growing facultative aerobes,microaerophiles and anaerobes. The use of the specially designed culturedish along with an oxygen reducing agent makes possible the design andfunction of a culture dish that utilizes some features of the BrewerLid, but overcomes its limitations and makes possible novel and improvedcharacteristics.

[0028] In this regard, the present invention is directed to aspecifically designed culture dish with a dish top or cover thatcontains a sealing Sing on the inside upon which the solid media surfacein the bottom dish rests when the dish is inverted to form a media-ringseal. The seal so formed traps the gas in the headspace between themedia surface and the inside of the dish top or cover. In addition, anoxygen reducing agent, such as a biocatalytic oxygen reducing agent, canbe incorporated into the media present in the culture dish together, insome instances, with a substrate which reacts with oxygen in the mediaand the headspace to create an environment suitable for growinganaerobic microorganisms.

[0029] The preferred biocatalytic oxygen reducing agent (see “A NovelApproach to the Growth of Anaerobic Microorganisms” of Adler, et al.,Biotechnol. Bioegn. Symp. 11, J. Wiley & Sons, New York, 1981, p. 533and U.S. Pat. No. 4,476,224 issued Oct. 9, 1984 to Adler entitled“Material and Method for Promoting the Growth of Anaerobic Bacteria”)utilized in the invention is comprised of oxygen scavenging membranefragments which contain an electron transport system which reducesoxygen to water in the presence of a hydrogen donor. These oxygenscavenging membrane fragments can be derived from the cytoplasmicmembranes of bacteria (U.S. Pat. No. 4,476,224) and/or from themembranes of mitochondrial organelles of a large number of highernon-bacterial organisms. Other known biocatalytic oxygen reducing agentssuch as glucose oxidase, alcohol oxidase, etc. can also be utilized.

[0030] The biocatalytic oxygen reducing agents suitable for use in theinvention are non-toxic to microorganisms. Being catalysts, they aredynamic and highly efficient at reducing the oxygen in the trappedheadspace in the specially designed culture dish. The biocatalyticoxygen

[0031] In this regard, the present invention is directed to aspecifically designed culture dish with a dish top or cover thatcontains a sealing ring on the inside upon which the solid media surfacein the bottom dish rests when the dish is inverted to form a media-ringseal. The seal so formed traps the gas in the headspace between themedia surface and the inside of the dish top or cover. In addition, anoxygen reducing agent, such as a biocatalytic oxygen reducing agent, canbe incorporated into the media present in the culture dish together, insome instances, with a substrate which reacts with oxygen in the mediaand the headspace to create an environment suitable for growinganaerobic microorganisms.

[0032] The preferred biocatalytic oxygen reducing agent (see “A NovelApproach to the Growth of Anaerobic Microorganisms” of Adler, et al.,Biotechnol. Bioegn. Symp. 11 J. Wiley & Sons, New York, 1981, p. 533 andU.S. Pat. No. 4,476,224 issued Oct. 9, 1984 to Adler entitled “Materialand Method for Promoting the Growth of Anaerobic Bacteria”) utilized inthe invention is comprised of oxygen scavenging membrane fragments whichcontain an electron transport system which reduces oxygen to water inthe presence of a hydrogen donor. These oxygen scavenging membranefragments can be derived from the cytoplasmic membranes of bacteria(U.S. Pat. No. 4,476,224) and/or from the membranes of mitochondrialorganelles of a large number of higher non-bacterial organisms. Otherknown biocatalytic oxygen reducing agents such as glucose oxidase,alcohol oxidase, etc. can also be utilized.

[0033] The biocatalytic oxygen reducing agents suitable for use in theinvention are non-toxic to microorganisms. Being catalysts, they aredynamic and highly efficient at reducing the oxygen in the trappedheadspace in the specially designed culture dish. The biocatalyticoxygen reducing agents use substrates that are commonly found inmicrobiological media and that are natural to microorganisms to effectthis reaction. The products produced from this reaction are also naturaland non-toxic to microorganisms. The use of the biocatalytic oxygenreducing agents makes possible the opening and closing of this dishseveral times and the agents continue to reduce the oxygen trapped inthe headspace after each occurrence.

[0034] The culture dish (“OxyDish™”) containing the oxygen reducingagent provides a means to work with microorganisms free of thecomplications and expense of anaerobic bags, jars, incubators, orchambers. Each dish is light in weight and is designed to be stackedwithout crushing the solid (agar or gelatin-based) medium in the lowerdishes in the stack. The dishes can be made of low cost materials,preferably plastic, are designed to be readily molded, are sterilizable,and preferably can be disposed after use. Because of the incorporationof a biocatalytic means of removing oxygen, an enlarged headspace ispossible. This enlarged headspace relieves the moisture condensationproblems encountered with the Brewer Lid.

[0035] Moreover, the dish top of the culture dish in certain embodimentsof the present invention, has a small dome or cavity designed to containan anaerobic gas (such as CO₂) generating pad or indicator strips toshow the anaerobic state within the headspace of the closed culturedish. A variation of this dish design provides for additional removal ofmoisture from the dish as needed by placing pores in the bottom of thedish base. This feature prevents the build-up of excess condensateinside the dish which leads to flooding of the agar media surface. Thepores are too small to let molten agar media flow out of the dish, yetthey provide an exit for moisture. An oxygen intruding into the dishthrough these pores must pass through the media containing the oxygenreducing agent. This intruding oxygen is removed before it can diffuseto the top layer of media or into the headspace where it would interferewith growth of anaerobic microorganisms.

[0036] The culture dish, i.e., “OxyDish™” of the present invention, isdesigned for automated preparation of agar or gelatin-based media platesnecessary for commercial production. When in the upright position, thedish can be readily filled with molten medium (such as a molten agar orgelatin-based media) without the sealing ring contacting the mediumsurface. When stored or used, the dish is placed into an invertedposition. In this position, a seal (i.e. a media-ring seal) is formed bythe contact of the sealing ring of the dish top with the media surfacecontained in the dish bottom when the media surface comes to rest on thesealing ring. This creates a headspace defined by the media surface, theinside wall of the sealing ring, and the inside top of the dish lid.

[0037] Furthermore, when the culture dish is utilized with the oxygenreducing agent such as a biocatalytic oxygen reducing agent, the oxygenreducing agent in the media reacts with the oxygen trapped in thatheadspace. This reaction renders the headspace sufficiently low inoxygen such that microorganisms affected by the presence of oxygen cangrow on the media surface typically within 24 to 48 hours when the dishis incubated at 35° C. to 37° C. in an aerobic incubator. Any oxygenthat intrudes into the dish around the media ring-seal or through theplastic is removed by the action of the reducing agent. The catalyticreducing agent facilitates the design and function of this dish.

[0038] The media suitable for use in the present invention includes anysolid type media which can be inverted to form a media ring-seal. Solidmedia generally consists of liquid media which have been solidified(“gelled”) with an agent such as agar or gelatin. Examples of otherknown suitable gelling agents include alginate, gellan gum (“Gelrite™”)and silica gel (“Pluronic Polyol F127™”). The solid type media is ofsuch a composition to support growth of anaerobes, microaerophiles andfacultative aerobes.

[0039] Further, the culture dish, i.e., “OxyDish™”, of the presentinvention, is designed in certain embodiments so that it can be stackedin a stable configuration. The dish top has a stacking ring thatinterlocks with the adjacent dish top below it. The dish bottom, whenthe assembled dish is inverted and placed in a sealed position, rests(i.e., nests) between the two adjacent dish tops. The functionality ofthe dish to establish and maintain an anaerobic environment is preservedand protected in the stack. The stackability of the culture dishincreases the efficient use of incubator space. Stackability is alsoimportant for the mechanized filling of these dishes and shipment ofdishes or of finished pre-made, plates to the microbiologist or enduser.

[0040] The culture dish of the present invention, simplifies handlinganaerobes by the microbiologist or laboratory technician. The culturedish, i.e., “OxyDish™”, can be opened and closed several times whilecontinuing to generate an anaerobic environment in the closed position.The specially designed culture dish significantly increases themicrobiologist's efficiency by reducing and simplifying the number ofmanipulations required to work with anaerobes. Furthermore, themicrobiologist can now treat each culture dish and its microbialcontents individually. This allows the microbiologist to make decisionsbased on his observations of each isolate or treatment, rather thanhaving to wait for the slowest growing isolate in a group of culturedishes present in a sealable jar, bag, etc. In addition, theself-contained, environmentally controlled culture dish provides asecure environment for the microbe inside.

[0041] The foregoing has outlined some of the most pertinent objects ofthe invention. These objects should be construed to be merelyillustrative of some of the more prominent features and applications ofthe intended invention. Many other beneficial results can be attained byapplying the disclosed invention in a different manner or by modifyingthe invention within the scope of the disclosure. Accordingly, otherobjects and a more detailed understanding of the invention may be had byreferring to the drawings, the detailed description of the invention andthe claims which follow below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The following is a brief description of the drawings which arepresented for the purposes of illustrating the invention and not forpurposes of limiting the same.

[0043]FIG. 1 is a cross-sectional view of a two-part culture dish forgrowing anaerobic microorganisms shown in separated or explodedrelation.

[0044]FIG. 2 is a cross-sectional view of the assembled culture dish ofFIG. 1 shown in what is generally referred to as a first or uprightposition.

[0045]FIG. 3 is a cross-sectional view of the assembled culture dishshown in what is generally referred to as a second or inverted position.

[0046]FIGS. 4A is a top view of a first component or bottom dish of theculture dish.

[0047]FIG. 4B is a side elevation view of the bottom dish.

[0048]FIG. 4C is a bottom view of the bottom dish.

[0049]FIG. 4D is an enlarged view of the side wall of the bottom dish.

[0050]FIG. 5A is a top view of the second component or dish top or coverof the culture dish.

[0051]FIG. 5B is a side elevational view of the dish top.

[0052]FIG. 5C is a bottom view of the dish top.

[0053]FIG. 5D is a sectional view of the dish top taken generally alongthe lines 5D-5D of FIG. 5C.

[0054]FIG. 6 is a side elevational view of two assembled culture dishesstacked vertically in an upright position.

[0055]FIG. 7 is a side elevational view of three assembled culturedishes stacked vertically in an inverted position.

[0056]FIGS. 8A and 8B are photographs exhibiting growth of anaerobicorganisms in the culture dish of the present invention.

[0057]FIG. 9 is a cross-sectional view of another preferred embodimentof a base or bottom dish of the culture dish assembly.

[0058]FIG. 10 is a cross-sectional view of another preferred embodimentof a lid or cover of the culture dish assembly.

[0059]FIG. 11 is an enlarged cross-sectional view of multiple culturedish assemblies stacked in a first orientation.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The culture dish of the present invention is designed to meet thestrict requirements of anaerobiosis while simplifying handling by themicrobiologist. As shown in the drawings, a culture dish 10 includes twoseparately configured parts. A first component or dish bottom 12receives a culture media 14 and a dish cover or top 16 defines a secondcomponent of the culture dish. The dish bottom and dish top selectivelycooperate to define a culture dish for growing microorganisms. Together,the dish bottom 12 and dish cover 16 define in a first position ororientation a covered Petri culture dish as shown in FIG. 2. This firstposition will be referred to as an upright position. When inverted (FIG.3), the dish bottom and dish cover alter their cooperative configurationto define a second position or orientation that forms an enclosedchamber or head space 80 in which anaerobic microorganisms 20 can becultivated.

[0061] The structural and functional details of the dish bottom 12 willbe described with reference to FIGS. 1-3, and more particularly withreference to FIGS. 4A-4D. The dish bottom is comprised of a generallyplanar base 22 and a circumferentially continuous side wall 24 extendinggenerally orthogonally from the base. For purposes of discussion only,the side wall will be described as extending upwardly from the base asillustrated in FIGS. 1, 2, and 4. It will be recognized, however, thatany directional description is merely for purposes of simplifying anunderstanding of the present invention.

[0062] In addition, the dish bottom 12 has a first or inner surface 26that faces inwardly over the base and side wall toward the cavitydefined by the cup-shaped arrangement of the base and side wall.Likewise, a second or outer surface 28 faces away from the cavity andencompasses the exterior surfaces of the base and side wall. The innersurface of the side wall is preferably divided by a lip 30 into firstand second portions 32, 34. The lip 30 can be optionally provided on theinner surface 26 of the side wall of the dish bottom and acts as a guidefor indicating the fill height of the culture media 14. The firstportion of the side wall defines an upper rim 36 and the second portion34 joins the upper rim to the base 22. As illustrated, the base and sidewall are shown as a one-piece construction such as a molded arrangement,although other equivalent configurations can be used without departingfrom the scope and intent of the invention.

[0063] The dish bottom 12 can be of any convenient dimension, and isusually circular so that this dimension is referenced as a diameter.Typically, the diameter of the dish bottom is about eight (8.0) tofifteen (15.0) cm. The depth of the dish bottom 12 defined by the heightof the side wall as it extends upwardly from the base can vary and isgenerally about 0.8 to 1.8 cm. In certain embodiments (FIG. 4C), thebase 22 of the dish bottom 12 can be divided into two, three, four ormore sections 38 by sectional dividers, grid markings or other indicia40 to enhance differential diagnostics of microorganisms (FIG. 4C).

[0064] The dish cover or top 16 (FIGS. 1 and 5A-5D) is sized to fit orconform over the dish bottom 12. The dish cover includes a top wall 50,first and second side walls 52, 54, and a seal ring 56. The top wall isdisposed at approximately mid-height of the outer side wall 52 forreasons which will be described in greater detail below. In a mannersimilar to the dish bottom, the side walls 52, 54 are disposed generallyorthogonal to the top wall and are themselves radially spaced apart by agap or recess 58. The side walls are joined along one end by aninterconnecting wall 60 to define an inverted, generally J-shapedconfiguration when the culture dish components are disposed in the firstor upright position (FIGS. 1 and 2).

[0065] The dish cover has a first or inner surface 70 that generallyfaces the dish bottom when the individual components of the culture dishare assembled. A second or outer surface 72 extends over the exterior ofthe dish cover.

[0066] The seal ring 56 projects outwardly or downwardly from the innersurface 70 of the top wall 50. The ring is circumferentially continuousand located along the radial periphery of the top wall. It alsointerconnects along its outer radial edge with the inner or second sidewall 54. The seal ring has a planar seal face 78 that cooperates withthe culture medium 14 in a dish bottom to define an anaerobicenvironment for growing microorganisms when disposed in an invertedposition (FIG. 3) and as will be described in greater detail below.

[0067] The recess 58 in the dish cover is defined between the first andsecond side walls. When the dish cover 16 is assembled with the dishbottom 12 and placed in an upright position (FIG. 2), the side wall 24of the dish bottom is received in the recess 58 of the dish cover. Therecess can vary in width depending upon the overall size andconfiguration of the dish cover and dish bottom.

[0068] Further, when the cover 16 is joined with the dish bottom 12 andpositioned in an upright position, the height of the side wall 52 of thedish cover is sufficient to keep the planar seal face 78 of the sealring 56 from contacting the medium surface 14. This allows the freshlypoured plate with molten agar, to cool and solidify before the mediasurface of the dish bottom 12 can rest on the seal ring in the dishcover 16 when the assembled culture dish 10 is inverted (FIG. 3). Thisfeature also provides a means for producing finished plates in acontinuous manner by mechanized means on a conveyor belt for large scalecommercial production while maintaining aseptic conditions.

[0069] When dish bottom 12 is filled with solidified media 14 and theassembled culture dish 10 is inverted, the solidified media surface willcome into contact with the seal ring 56 of the dish cover 16 forming amedia-ring seal along the planar seal face 78 (see FIGS. 3 and 7). Inthis inverted configuration of the culture dish, an oxygen reducingagent present in the media 14 will remove all of the oxygen that istrapped in the head space of the enclosed chamber 80 formed between thesurface of the solidified media and the inner surface 70 of dish cover.The assembled culture dish can be incubated aerobically in the invertedposition while producing an internal anaerobic environment for thegrowth of anaerobic microorganisms 20.

[0070] In addition, in certain embodiments of the invention, a raisedarea, dome or cavity 82 is present in the dish cover 16 (FIG. 1).Specifically, projecting outward from the top wall 50 of the dish coveris a dome 82 designed to contain an anaerobic gas (CO₂, etc.) generatingpad or an indicator strip (not shown). The dome 82 is comprisedessentially of a dome side wall 84 and a dome top wall 86, although acircular dome is the more preferred embodiment. It is understood bythose skilled in the art that domes or cavities of alternative shapesand sizes can be utilized with equal success.

[0071] In accordance with the illustrated embodiment, strengthening ribs90 are peripherally spaced along side wall 52. The ribs are preferablyequally spaced about the circumference of the dish cover and protruderadially outward from the exterior surface of the side wall. The ribsprovide additional rigidity and strength to the dish cover which isparticularly helpful when the culture dishes are stacked in either theupright or inverted positions as shown in FIGS. 6 and 7.

[0072] The dish cover 16 can be of any convenient diameter so long as itmates with the dimensions of the dish bottom 12. Typically, the dishcover 16 is approximately nine (9.0) cm to sixteen (16.0) cm indiameter. The seal ring 56 can be of any desired diameter and isgenerally about seven (7.0) cm to fourteen (14.0) cm in diameter and iscentrally positioned relative to the side wall 52 of the dish cover. Theoverall radial dimension of the seal ring 56 can vary with a preferredradial dimension being about two-tenths (0.2) cm to one-half (0.5) cm.

[0073] Moreover, in some embodiments, the base 22 of the dish bottom 12contains an indicator ring 100 (FIG. 4A). The area or annulus 102between the indicator ring 100 of base and the side wall 24 identifiesthe area on the media surface 14 that the seal ring 56 will occupy whenthe assembled culture dish 10 is inverted. This area is not to be usedfor culturing microorganisms. Microbes present in this area will swarmaround the seal ring 56 when the dish top is placed in contact with theculture media.

[0074] The difference in the height of the side wall 52 of the dishcover in relation to the height of the side wall 24 of the dish bottomcan also vary, with a height differential of about one-half (0.5) cmbeing preferred. The fill height 110 in FIG. 1 and FIG. 4D is thedistance from the base 22 of the dish bottom 12 to the surface level ofthe culture media 14 and is variable. Typically, this height can betwo-tenths (0.2) cm to four-tenths (0.4) cm. The dimension from the topof the culture media 14 surface, which is generally indicated by theinner lip 30 to the top edge of the dish bottom is (D) and is determinedby the relationship D=A−C. (see FIG. 4D, wherein (A) is the total heightof the side wall 24 of the dish bottom 12 and (C) is the fill height ofthe culture media).

[0075] The seal ring 56 inside the dish cover extends a distancedownward from the dish cover 16 equal to (E) in FIG. 5B which isdetermined by the relationship E=B−(Cmax+0.1 cm), where (B) is the totalheight of the side wall 52 of the dish cover and (Cmax) is the maximumfill height of the culture media 30. This assures that the seal ring 56clears the culture medium surface by approximately one-tenth (0.1) cmwhen the dish bottom 12 is filled to its maximum level and the assembledculture dish 10 is in its upright position (FIG. 2).

[0076] The distance between the top edge of the side wall 24 of the dishbottom and the upper extreme of the inner surface 70 of the dish cover16 when the culture dish is oriented in the upright position is (F) inFIG. 6. In a preferred embodiment, F is determined by F=B−A, where (B)is the total height of the side wall 52 of the dish cover 16 and (A) isthe total height of the side wall 24 of the dish bottom 12.

[0077] The depth of the headspace or enclosed chamber 80 below the mediasurface 38 formed when the assembled culture dish is inverted to form amedia-ring seal is determined by the dimension (G) in FIG. 7. Thisdimension can vary depending on the size of the dish top 16, buttypically ranges between two-tenths (0.2) cm to one-half (0.5) cm. Thedimension (H) in FIG. 7 is the height of the top wall from an upper edgeof the dish cover. It is determined according to the followingrelationship H=E−G, where (E) is the height of the inner side wall 54 ofthe dish cover and (G) is the height of the headspace 80.

[0078] The dish cover preferably includes one or more cut-out areas 112(FIG. 5B) in a portion of the side wall 52. These cut-out areas 112facilitate the grasping and separation of the dish bottom 12 from thedish cover in an assembled culture dish 10. The cut-out areas may bevariably or constantly spaced from each other in the side wall 52 of thedish cover. As shown, one preferred arrangement has two cut-out areas112 in the side wall 52 that are equidistant from each other. Likewise,the particular configuration of the cut-out areas may vary withoutdeparting from the scope and intent of this invention.

[0079] Moreover, in the preferred embodiment of the invention, theassembled culture dishes 10 are designed to be stacked one on top ofanother. A dish bottom 12 of one assembled culture dish is nestledbetween stacked dish covers 16 (see FIG. 6) in the upright positions. Inthis regard, each dish cover has a stacking ring or protruding rib 120around the upper edge of the dish cover (FIG. 1). While the diameter ofthe stacking ring 120 can vary, it is generally about one-half (0.5) mmto one (1.0) mm less than the overall peripheral diameter of the dishcover 16. This provides an outer radial ledge 122 upon which the bottomedge of the side wall 52 of an adjacent dish cover rests when placedeither in an upright (FIG. 6) or inverted position (FIG. 7). Theprojection of the stacking ring 120 is preferably about one-half (0.5)mm to one and one-half (1.5) mm in height. The stacking ring 120prevents an adjacent dish cover from sliding laterally and upsetting thestacked arrangement (see, for example, FIGS. 6 and 7).

[0080] Similarly, the stacking ring 120 on the dish cover 16 radiallycontains or nests an adjacent dish bottom 12 when stacking is desired inan upright position (see FIG. 6). The stacking ring 120 defines a radialinner ledge 124 to impede slide-out of the enjoining dish bottom 12. Thestacking ring is preferably one-half (0.5) mm to (1.5) mm in width.

[0081] The minimum fill height (Cmin) to which the dish bottom 12 can befilled with culture media 14 and have the media surface rest on the sealring 56 when the dish is in an inverted position is determined byCmin=A−E, wherein (A) is the total height of the side wall 24 of thedish bottom 12 and (E) is the height of the inner side wall 54 of thedish cover. If the fill height of the culture media 14 is below thislevel, then the upper rim of dish bottom 12 rests in contact with theinner surface 70 of the dish cover 16 rather than the media surface 14resting on the seal ring 56 of the dish cover when the assembled culturedish is inverted. In this situation, there is no seal formed between theseal ring 56 and the medium surface 14. The sealed headspace 80 is notformed. This condition renders the assembled culture dish 10 useless forone designed purpose of the culture dish which is to provide aself-contained environment for the isolation and growth ofmicroaerophiles and anaerobes.

[0082] A variant of the culture dish contains one or more perforationsor pores 132 in the dish bottom 12 for the purpose of controllingmoisture inside the headspace 80. The sizes of the pores 132 can varybut are usually about one-tenth (0.1) cm to three-tenths (0.3) cm indiameter. The number of pores 132 can vary from one (1) to eighty (80)or more and their location can be grouped or evenly spaced. The poresmay be covered with an adhesive film (not shown) such as Mylar™ whichretards the passage of oxygen and can be sterilized in place when thedish is sterilized. This film can be removed after the culture dish 10is filled and before it is incubated. The pores provide a means toreduce the water content of the media during incubation in a controlledmanner. This reduces the condensate that forms inside the assembledculture dish 10. Any oxygen infiltrating into the assembled culture dish10 through these pores 132 must pass through the media 14 to get to themedia surface where the microbes 20 are planted. The media 14 containsthe biocatalytic oxygen reducing agent and optionally one or moresubstrates that removes the oxygen before it can reach the surface bythis route.

[0083] The culture dish 10 is designed to be easily manufactured byknown injection molding techniques. The dish top 16 and dish bottom 12have no features that prevent them from being ejected from a mold. Thematerials of construction can vary but are preferably polystyrene,polycarbonate, or polystyrene-acrylonitrile. These are clearthermoplastics that are inexpensive, easy to mold, sterilizable byethylene oxide or radiation, resilient to handling and resistant tochemical substances used in microbiological media. Styrene-acrylonitrilehas the lowest oxygen permeability of the three thermoplasticsmentioned. All of the parts are preferably transparent to permitobservation of the anaerobic culturing process. However, pigments ordyes may be added to the polymeric materials in order to producedifferent shades or colors. Further, as it is understood by thoseskilled in the art, ultra-violet light absorbers and other additives canbe added to produce culture dishes having the properties desired by theend user.

[0084] The assembled culture dish 10 can be opened by one of threemethods:

[0085] A) The assembled and sealed culture dish 10 is placed upright ona bench top. The dome 82 on the dish top 16 is depressed. The flexing ofthe dish top 16 causes the media-ring seal to part releasing andallowing the dish bottom 12 to come to rest on the bench top.

[0086] B) The assembled and sealed culture dish 10 is gently struck ontothe bench surface. This action breaks the media-ring seal which in turnreleases the dish bottom 12 and allows the dish bottom 12 to come torest on the bench top.

[0087] C) The assembled and sealed culture dish 10 is placed upright ona bench top. The side walls 52 of the dish top 16, between cut-outareas, are gently flexed. This action causes the media-ring seal to partand releases the dish bottom 12 to come to rest on the bench top.

[0088] The media-ring seal can be reformed simply by placing the dishtop 16 over dish bottom 12 and re-inverting the assembled culture dish10. Gravity will cause the dish bottom 12 containing the solidifiedmedia to come into contact with the seal ring. The substrate and/oroxygen reducing agent present in the media 14 will once again remove allof the oxygen trapped in the head space 80.

[0089] A second preferred embodiment is illustrated in FIGS. 9-11. Wherepossible, like numerals will identify like components with a primedsuffix, while new components will be identified with a new numeral.Moreover, it will be appreciated that the culture dish assembly of thissecond preferred embodiment operates in substantially the same manner,i.e., having first and second orientations in which the dish componentscooperate with one another.

[0090] A first dish or base 12′ has a substantially planar wall 22′ anda side wall 24′ extending therefrom. The side wall has a slight outwardtaper as it proceeds outwardly from the wall 22′. A culture medium oragar 14′ is received in the cup-shaped first dish component (FIG. 11).As described above, the fill height of the culture medium is importantso that an effective seal can be achieved between the dish componentsand the second or inverted orientation. A rib 150 extends outwardly fromthe generally planar base in a direction generally opposite that of theside wall 24′. In the preferred arrangement, the rib 150 iscircumferentially continuous and is radially dimensioned and located toseat on a terminal end of the second leg of the second dish component orlid, as will be described in greater detail below.

[0091] The second dish component 16′, also referred to as a cover, lid,or top, is sized to fit and conform over the dish bottom. It includes awall 50′ that is substantially planar and first and second side walls52′, 54′. Radially interposed between the planar wall 50′ and the secondleg 54′ is the seal ring 56′ defined by a planar, annular seal face. Aswill be appreciated from a review of FIG. 11, it is apparent that thefirst and second legs 52′, 54′ of the lid have tapered conformations. Inparticular, they define a generally inverted U-shaped configuration. Thesecond leg 54′ tapers radially outward from its interconnection with theseal region 56′ to an interconnecting portion 152. The interconnectingportion 152 has a radial dimension that supports the rib 150 extendingfrom the first dish. A raised protrusion 154 assists in centering a dishbase when disposed in stacked relation on the lid of an adjacent culturedish assembly. Moreover, a support region 156 is disposed radiallyoutward of the protrusion and is adapted to receive the terminal end 158of the first leg of an adjacent lid. In this manner, assembled dishcomponents in the first orientation can be stacked one atop the other(FIG. 11). The side wall 24′ of the base is then received between thediverging first and second legs 52′, 54′ of the lid. The degree of taperis sufficiently controlled so that region 152 is located for supportingthe rib 150 of an adjacent base and region 156 of the interconnectingportion provides suitable support for the first leg of the lid.Moreover, the protrusion 154 serves a centering function to facilitatestacking of the dish components.

[0092] In this arrangement, the circumferential side wall defined by thefirst leg 52′ of the lid is also preferably circumferentiallycontinuous. Although the cutouts may facilitate handling of the lid,warpage could occur during the molding operation, thus it will beappreciated that for more precise control of the manufactured component,a circumferentially continuous side wall may be desired in someinstances.

[0093] Although not illustrated, it will be appreciated that the dishcomponents of the second preferred embodiment also cooperate in thesecond orientation, or inverted relation in a manner similar to thatdescribed above. Thus, the seal face 56′ will suitably seat or sealagainst the surface of the agar 14′. The space between the first andsecond legs 52′, 54′ of the lid allows the side wall 24′ of the base tomove inwardly toward the interconnecting portion between the legs. Thismovement is stopped by engagement between the seal face and the agar todefine a sealed headspace between the culture medium and the generallyplanar wall 50′.

[0094] While the culture dish of the invention has been shown anddescribed herein as being particularly adapted for use in circular form,it is not desired or intended to thus restrict the scope and utility ofthe improvements by reason of such specific embodiments since theapparatus may be of various shapes and sizes without departing from theinvention. In addition, it is also contemplated that certain specificdescriptive technology used herein shall be given the broadest possibleinterpretation consistent with the disclosure.

[0095] The biocatalytic oxygen reducing agents suitable for use in theinvention include known biocatalytic oxygen reducing agents such asglucose oxidase and catalase and the oxygen scavenging bacterial cellmembrane fragments disclosed in U.S. Pat. No. 4,476,224 entitled“Material and Method for Promoting the Growth of Anaerobic Bacteria”,issued Oct. 9, 1984 to Howard I. Adler, Oak Ridge, Tenn., one of theco-inventors of the present invention. The '224 patent is incorporatedherein by reference.

[0096] The '224 patent is directed to a method of removing dissolvedoxygen from a nutrient medium for anaerobic bacteria through the use ofsterile membrane fragments derived from bacteria having membranes whichcontain an electron transport system which reduces oxygen to water inthe presence of a hydrogen donor in the nutrient medium. It is knownthat a great number of bacteria have cytoplasmic membranes which containthe electron transport system that effectively reduces oxygen to waterif a suitable hydrogen donor is present in the medium. Some of thebacterial sources identified in the '224 patent include Escherichiacoli, Salmonella typhimurium, Gluconobacter oxydans, and Pseudomonasaeruginosa. These bacterial membranes have been highly effective inremoving oxygen from media and other aqueous and semi-solidenvironments.

[0097] The same oxygen reducing effects produces by the cell membranefragments from the bacterial sources indicated above, are also presentin the membrane of mitochondrial organelles of a large number of highernon-bacterial organisms. More particularly, a great number of fungi,yeasts, and plants and animals have mitochondria that reduces oxygen towater, if a suitable hydrogen donor is present in the medium. Some ofthe sources of oxygen reducing membranes from these mitochondria are:beef heart muscle, potato tuber, spinach, Saccharomyces, Neurospora,Aspergillus, Euglena and Chlamydomonas. The process of producing theuseful mitochondrial membrane fragments involves the following steps:

[0098] 1. Yeast, fungal cells, algae and protozoa, having mitochondrialmembranes containing an electron transfer system which reduces oxygen towater, are grown under suitable conditions of active aeration and atemperature which is conducive to the growth of the cells, usually about20° C. to 45° C. in a broth media. Alternately, mitochondria may beobtained from the cells of animal or plant origin.

[0099] 2. The cells are collected by centrifugation or filtration, andare washed with distilled water.

[0100] 3. For the preparation of crude mitochondrial membrane fragments,a concentrated suspension of the cells is treated to break up the cellwalls and mitochondria. This is accomplished by known means, forexample, by ultrasonic treatment or by passing the suspension severaltimes through a French pressure cell at 20,000 psi.

[0101] 4. The cellular debris is removed by low speed centrifugation orby microfiltration (cross-flow filtration).

[0102] 5. The supernatant or filtrate is subjected to high speedcentrifugation (175,000× g at 5° C.) or ultrafiltration.

[0103] 6. For the preparation of material of higher purity, the cells ofstep 2 are suspended in a buffer containing 1.0 M sucrose and aretreated by means which break up the cell walls or membranes but leavethe mitochondria intact. This is accomplished by known means, forexample, by ultrasonic treatment, passage through a French pressure cellat low pressure, enzymatic digestion or high speed blending with glassbeads.

[0104] 7. The cellular debris from step 6 is removed by differentialcentrifugation or filtration.

[0105] 8. The supernatant or retentate from step 7 is passed through aFrench Press at 20,000 psi to break the mitochondria into small pieces.

[0106] 9. Mitochondria debris from step 7 is removed by centrifugationat 12,000× g for approximately 15 minutes or by microfiltration.

[0107] 10. The supernatant or filtrate from step 9 is subjected to highspeed centrifugation (175,000× g at 5° C.) or ultrafiltration.

[0108] 11. The pellet or retentate from step 5 (crude mitochondrialfragments) or the pellet or retentate from step 10 (purifiedmitochondrial membrane fragments) are resuspended in a buffer solutionat a pH of about 6.0 to about 8.0. A preferred buffer solution is 0.02 Msolution of N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid(HEPES).

[0109] 12. The membrane fragments in the buffer solution are then passedunder pressure through a filter having openings of about 0.2 microns.

[0110] 13. The suspension is then stored at about −20° C. for later useor it may be freeze dried.

[0111] Furthermore, while many solidified medium do not require theaddition of a hydrogen donor in order for the enzyme system present inthe membrane fragments to reduce the oxygen present in the product towater, when synthetic medium or medium failing to contain a hydrogendonating substance are utilized, the addition of a hydrogen donor (i.e.,an organic substrate) may be necessary in order for the membranefragments to perform their oxygen removing functions. Suitable hydrogendonors are lactic acid, succinic acid, alpha-glycerol phosphate, formicacid, malic acid, and where available, their corresponding salts.

[0112] The present invention is further illustrated by the followingexamples. It is to be understood that the present invention is notlimited to the examples, and various changes and modifications may bemade in the invention without departing from the spirit and scopethereof.

EXAMPLES Example 1 Growth of Anaerobic Microorganisms Using the CultureDish, i.e., “OxyDish™” of the Present Invention and a BiocatalyticOxygen Reducing Agent

[0113] Nutrient agar is supplemented with sodium formate (15 mM), sodiumsuccinate (30 mM), sodium lactate (45 mM) and cysteine (0.025 g/100 ml).A biocatalytic oxygen reducing agent, EC-Oxyrase® (Oxyrase, Inc.,Mansfield, Ohio) is added to cooled (45° C. to 50° C.) but moltensterile medium to give a final concentration of 5 units/ml. 20 ml of theabove mixture is soon introduced into the bottom part of a culture dish,i.e., “OxyDish™”. The top part of the culture dish, is placed over thefilled bottom part to prevent contaminants from entering the dish. Theagar in the bottom part cools to ambient temperature and solidifies. Thecovered dish is left standing to permit excess moisture to escape. Atthis point the dish may be sealed by inverting it to bring the agarsurface in the dish bottom into contact with the ring inside the dishtop.

[0114] A suspension of anaerobic microorganisms is spread on the surfaceof the agar medium that contains the biocatalytic oxygen reducing agentand its substrates. The dish is sealed by inverting it. The dish is thenplaced into an aerobic incubator at 35° C. to 37° C. for 24 to 48 hours.Several dishes are stacked to form a stable column of dishes.

[0115] Assembled dishes can be handled and viewed at any time withoutbreaching the seal and losing the anaerobic environment inside thetrapped headspace. In this way, a particular culture dish, i.e.,“OxyDish™” can be selected at the earliest time when the microbialisolate has grown sufficiently for selection.

[0116] Using this technique with the culture dish, i.e., “OxyDish™” anda biocatalytic oxygen reducing agent, the following microorganisms havebeen grown:

[0117]Clostridium tertium, C. difficile, C. perfringens, C. cadaveris,C. acetobutylicum, Bacteroides thetaiotaomicron, B. fragilis, B.distasonis, Escherichia coli, Fusobacterium varium, F. mortiferum, F.necrophorum, Peptostreptococcus magnus, P. anaerobius, P. nigra, P.intermedius, Lactobacillus casei, L. acidophilus, Eubacterium lentum,Bifidobacterium breve, and Streptococcus fecalis.

Example 2 Measurement of Oxygen Depletion in the Headspace of theCulture Dish Effect of the Present Invention by the Biocatalytic OxygenReducing Agent

[0118] A hole is drilled in the base of the culture dish, i.e.,“OxyDish™” and a gas tight septum is inserted. The base is then filledwith 20 ml of agar containing a biocatalytic oxygen reducing agent. Thebottom is sealed to the top by inverting the assembled dish andincubating it at 37° C. Periodically, 50 ul samples of the gas in theheadspace of the dish are sampled by inserting the tip of a 100 ul gastight Hamilton syringe through the septum in the base of the dish. Thesesamples are introduced into an Oxygen Sensor (IT Corporation) and theconcentration of oxygen remaining in the headspace is determined. Usingthis method it has been determined that all measurable oxygen, less than10 pp billion, is removed from the head space in two to eight hoursdepending on the concentration and configuration of the biocatalyticagent used. It has also been determined that the dish can be opened,resealed and, after a suitable incubation period, the head space againbecomes anaerobic.

Example 3 Multiple Opening and Closing of the Culture Dish of thePresent Invention and Reestablishment of Anaerobic Environment

[0119] A culture dish, i.e. “OxyDish™” of the present inventioncontaining a nutrient agar and the biocatalytic agent is streaked withan anaerobic organism (Bacteroides thetaiotaomicron or B. fragilis)covering two quadrants of the dish. After 24 hours of incubation at 37°C. the dish is opened, growth of the anaerobe is observed and a smallquantity of organism is streaked on the third quadrant of the dish. Thedish is resealed and after 24 hours of incubation at 37° C., the dish isreopened and growth is observed in the third quadrant. A small amount ofgrowth from the third quadrant is streaked on the fourth quadrant. Thedish is resealed and after a 24 hour incubation at 37° C. it is reopenedand growth is observed in the fourth quadrant of the dish.

Example 4 Rapid Anaerobiosis of the Agar Layer Containing a BiocatalyticOxygen Reducing Agent as Indicated by Methylene Blue.

[0120] An agar medium was made that contained water, 50 mM sodiumlactate, and 2.5 mg/ml of methylene blue. In the oxidized statemethylene blue is blue in color. In the reduced state it is colorless.The agar was melted and cooled to 45° C. It is blue in color.EC-Oxyrase® is added at 5 units/ml and 20 ml is delivered to the bottompart of a culture dish, i.e., “OxyDish™”. As soon as the agar hassolidified, about 5 minutes later, the culture dish, i.e., “OxyDish™” issealed by inverting it. At this time the agar layer is blue in color.The culture dish, i.e., “OxyDish™” is incubated at 37° C. and observedperiodically. Soon after sealing the culture dish, i.e., “OxyDish™”, theagar layer begins to lighten in color. Within 30 minutes to 45 minutesof being put into the incubator, the medium is nearly white inappearance, but with a light blue tinge of color. By 60 minutes ofincubation the agar layer is white, which indicates that the agar layeris anaerobic shortly after the addition of EC-Oxyrase® to the medium.

Example 5 Use of Methylene Blue Strip to Indicate Anaerobiosis in theCulture Dish

[0121] A small rectangular piece of filter paper impregnated withmethylene blue at an alkaline pH is fixed to the inside of the dome inthe top of the culture dish, i.e., “OxyDish™”. The dish bottom containsnutrient agar and a biocatalytic oxygen reducing agent. The dish issealed by inverting it thereby causing the agar surface to rest on thering. After incubation at 37° C. for 8 hours or more, the blue colordisappears from the filter paper. This indicates that the headspace ofthe culture dish, i.e., “OxyDish™” has become anaerobic.

Example 6 Use of Glucose Oxidase and Catalase as the Biocatalytic OxygenReducing Agent

[0122] Sterile Nutrient agar (Difco), supplemented with 1%i glucose, iscooled to 45° C. and 1 unit of sterile filtered glucose oxidase/ml, and1 unit of sterile filtered catalase (Sigma Biochemicals, 1994 Catalog, p221 and p 478) is added. 20 ml of this medium is deliver into a culturedish, i.e., “OxyDish™”. After the agar has solidified, a small quantityof Bacteroides fragilis is streaked on the surface of the agar and thedish is sealed by inverting it. After 48 hours of incubation at 37° C.,growth of the anaerobic microorganisms is observed on the surface of theagar medium.

Example 7 Use of a Filter Pad with Carbonate to Generate CO₂ in theHeadspace

[0123] A piece of filter paper saturated with a 1% sodium bicarbonatesolution and then dried is fixed to the inside of the dome in theculture dish, i.e., “OxyDish™” top. This filter paper is then covered bya 0.2 u membrane filter. The dish bottom is filled with 20 ml ofNutrient Agar (Difco) and a biocatalytic oxygen reducing agent,EC-Oxyrase® at 5 units/ml and substrates. The agar surface is inoculatedby streaking with a small amount of Clostridium acetobutylicum, amicroorganism that requires CO₂ for rapid colony development.Immediately before sealing the dish, one drop of 0.1 N HCl is placed onthe membrane filter. The dish is sealed by inverting it and placed intoa 37° C. aerobic incubator. After 24 hours of incubation, growth of C.acetobutylicum can be observed indicating that CO₂ was released from thesodium bicarbonate impregnated filter paper into the headspace of theculture dish, i.e., “OxyDish™”.

Example 8 Relief of Moisture Through Pores in the Dish Bottom

[0124] Seventy-six holes of different sizes (large: 0.101 inch, medium:0.086 inch, and small: 0.059 inch) are drilled into a culture dishbottom. The dish is filled with 40 ml of 1.5% agar. A standard dishcover or a Brewer Lid is fitted onto each dish bottom. The complete dishis weighed. The covered dish is incubated at 37° C. and the weighed attimed intervals. The loss of weight is taken as due to the loss ofmoisture, since the solidified agar is 98.5% water by weight. Therelative weight loss, net of control weight loss, is as follows: PoreSize 24 hrs. 48 hrs. Small  6% 11% Medium  9% 14% Large 13% 24%

[0125] This shows that the drying of the agar layer can be controlledduring incubation by the number and size of pores put into the dishbottom. For all size holes, the molten agar did not escape through theseholes. The Brewer Lid covered dishes exhibited no moisture build upwithin the trapped headspace for those dishes with holes in them.

Example 9 Comparison of Present Invention With Standard Methods forGrowing Anaerobes

[0126] The inoculum is prepared by selecting colonies of particularmicrobes from Wilkens-Chalgren blood agar plates. A loopful of growth issuspended in Brucella broth to a density of about 1.5×10⁸ colony formingunits per mL. The suspended microorganisms are put into wells of areplicator block. Sterile replica or pins are dipped into the wells ofthe block. The replicator pins are stamped onto the surface of agarmedium (Wilkens-Chalgren blood agar). Each pin is calibrated to deliverabout 1×10⁵ colony forming units per spot. This procedure is repeated toinoculate a controlled pattern of spots onto agar plates containingincreasing amounts of antibiotic. Appropriate control plates, that donot contain antibiotics, are included. After a short time, the spotsdry, the culture dish, i.e. the OxyDish, is sealed and incubatedaerobically. Standard plates, not containing the oxygen reducing agent,i.e. Oxyrase®, and substrates, are incubated in anaerobic jars orchambers. After 48 hours of incubation at 35° C. the plates are scoredfor growth. The presence of growth on a plate containing antibioticindicates that the particular microbe is resistant to the level ofantibiotic in that plate. In this way, one can determine the antibioticsusceptibility profile of a large number of microbial specimens.

[0127] With respect to the rate and intensity of growth of a number ofdifficult anaerobes using the culture dish of the present invention andthe brocatalytic oxygen reducing agent compared to the standard methods,it was found that anaerobic microbes grew faster and to a greaterdensity with the present invention compared to standard anaerobicmethods. As shown below, this observation was particularly noticeablefor difficult to grow anaerobes. Microbe Standard Method OxyDish MethodClostridium dificil 1+ 3+ Clostridium 4+ 4+ perfringens Clostridium 1+3+ cadaveris Bacteroides 3+ 4+ rhetaiotaomicron Bacteroides 3+ 4+distasonis Fusobacterium 2+ 4+ varium Fusobacterium 2+ 4+ mortiferumFusobacterium 1+ 3+ necrophorum Peptostreptococcus 2+ 3+ magnusPeptostreptococcus 1+ 3+ anaerobius Peptostreptococcus 1+ 3+ negraBifidobacterium 1+ 3+ breve Prevotella 2+ 3+ intermedia

[0128] The invention has been described with reference to the preferredembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such alterations and modifications insofar as they come within thescope of the claims and the equivalents thereof.

1. A culture dish assembly comprising: a first dish having a wall and aside wall extending therefrom to define a cavity; a second dish having awall and a side wall extending therefrom that cooperate with the firstdish to form a non-sealed assembly in a first orientation of the firstand second dishes and a closed headspace in a second, invertedorientation of the first and second dishes.
 2. The assembly of claim 1wherein the wall of the first dish has a substantially circularconfiguration.
 3. The assembly of claim 2 wherein the side wall of thefirst dish extends outwardly in a substantially perpendicular directionfrom a periphery of the circular wall.
 4. The assembly of claim 3wherein the side wall of the first dish extends in a first directionfrom the circular wall.
 5. The assembly of claim 1 wherein the wall ofthe second dish has a substantially circular configuration.
 6. Theassembly of claim 5 wherein the side wall of the second dish has agenerally U-shaped configuration in cross-section defining an annularrecess dimensioned to receive at least a portion of the side wall of thefirst dish therein.
 7. The assembly of claim 6 wherein the side wall ofthe second dish includes first and second legs disposed in generallyparallel relation, and the first leg is longer than the second leg anddisposed radially outward thereof.
 8. The assembly of claim 7 whereinthe second dish further includes an annular, circumferentiallycontinuous seal disposed radially inward of the side wall and isdisposed in a plane intermediate a terminal end of the first leg and thewall.
 9. The assembly of claim 8 wherein the side wall of the first dishhas a height less than the first leg of the first dish so that theannular seal is spaced from the wall of the first dish in both the firstand second orientations of the first and second dishes.
 10. The assemblyof claim 8 wherein the side wall of the second dish is circumferentiallycontinuous.
 11. The assembly of claim 1 wherein the wall of the firstdish is substantially planar.
 12. The assembly of claim 1 wherein thewall of the second dish is substantially planar.
 13. The assembly ofclaim 1 wherein the side wall of the first dish tapers radially outwardas it extends from the wall.
 14. The assembly of claim 1 wherein theside wall of the second dish includes first and second legs defining anannular recess therebetween, the first leg tapering radially outward asit extends substantially perpendicular from the wall.
 15. The assemblyof claim 14 wherein the second leg tapers radially outward as it extendssubstantially perpendicular from the wall.
 16. The assembly of claim 14wherein the wall of the first dish includes a rib extending outwardlytherefrom and radially dimensioned to seat on a terminal end of thesecond leg of the second dish.
 17. The assembly of claim 16 wherein aterminal end of the side wall of the first dish is dimensioned forradial receipt between the first and second legs of the second dish. 18.The assembly of claim 14 wherein the first and second legs of the seconddish are connected by an interconnecting portion having a raisedprotrusion for locating the wall of the first dish when the first andsecond dishes are disposed in the first orientation.
 19. The assembly ofclaim 14 wherein the first and second legs of the second dish areconnected by an interconnecting portion that has a radial dimensionadapted to support the wall of a first dish and a terminal end of asecond leg of an adjacent second dish in a first orientation of thedishes.
 20. The assembly of claim 19 wherein the interconnection portionincludes a raised protrusion separating regions on which the wall of afirst dish and a terminal end of a second leg of an adjacent second dishare supported in a first orientation of the dishes.